臺灣博碩士論文加值系統

免疫療法在癌症治療當中扮演相當重要的角色。早期的研究指出，以腫瘤細胞為基礎所製成的疫苗，能夠誘導具有腫瘤特異性的T細胞反應來引發系統性的抗腫瘤免疫力，且巨噬細胞聚落刺激因子（GM-CSF）也被廣泛地作為佐劑使用。在先前的研究中，我們觀察到在小鼠體內接踵DNA疫苗並嵌有GM-CSF序列修飾（codon-optimized）的質體，能誘發強力的抗體與毒殺性T細胞反應。然而對於多次施打疫苗是否會引起有效的抗腫瘤免疫性，仍然是個未知數。根據近期的研究發現，多次施打由GM-CSF分泌能力細胞所製成的疫苗，會因為調節性T細胞（Treg）的表現量增加而限制了疫苗的效價。本篇我們利用具有GM-CSF高分泌能力細胞製成的疫苗，並以不同次數的方式施打於小鼠，來觀察小鼠體內之調節性T細胞與免疫調控的關係。經輻射照射後的細胞將會被注射至小鼠皮下，並分別在施打一次、三次及五次後，進行調節性T細胞之免疫螢光染色與淋巴球再刺激試驗。根據實驗結果發現，經多次施打疫苗後，小鼠系統性的免疫反應中之調節性T細胞的表達量增加；且在HPV E7專一胜肽刺激後，小鼠體內對於GM-CSF序列修飾後之細胞製成疫苗，在僅施打一次時，其具丙型干擾素（IFN-γ）分泌能力的CD8+ T細胞表現量為最高。本篇實驗結果觀察到，多次施打高劑量的GM-CSF疫苗並無法誘導有效的抗腫瘤免疫反應。

Immunotherapy is playing a critical role in the treatment of cancers. It has been reported that tumor-cell based vaccine can induce tumor-specific T cell responses to prime systemic antitumor immunity, and GM-CSF is now widely used as an adjuvant in immunotherapy protocols. Our previous studies have shown co-immunization of plasmids encoded codon-optimized GM-CSF sequence with DNA vaccine would induce potent antibody and cytotoxic T lymphocyte responses. However, weather vaccination strategies that given multiple times would induce the effective antitumor immunity remain unclear. According to recent studies, multiple vaccinations of GM-CSF secreting tumor resulted in limiting the efficacy of vaccine by the increase level of regulatory T (Treg) cells. In our study, we used codon-optimized GM-CSF-secreting cell-based vaccine, which expressed high level GM-CSF, combined with multiple vaccination strategies to investigate the immune modulation by regulatory T (Treg) cells. Mice were subcutaneously injected with 106 irradiated cells each site, and we sacrificed mice after one-, three-, and five-times vaccinations for Treg cell staining and re-stimulation assay. The results showed increased Treg cells in systemic immune responses following multiple vaccinations. Moreover, after restimulated with E7-specific peptide, the immune cells from the mice which had one-time vaccination with codon-optimized GM-CSF-secreting cell-based vaccine showed the highest percentages of IFN-γ-secreting CD8+ T cells compared to three- or five-times vaccinations. Taken together, our data suggested that high-dose GM-CSF vaccine combined with multiple vaccinations would not induce the effective antitumor immune responses.

CHAPTER I Introduction………………………………………………1
I.1 Human papillomavirus (HPV)…………….……………………….1
I.1.1 HPV genome…………………………………………….…...1
I.1.2 HPV types………………………………………………..…..1
I.1.3 HPV viral oncogenes…………………………………………1
I.1.4 HPV vaccine……………………………………………….....2
I.2 Granulocyte-macrophage colony-stimulating factor (GM-CSF)…..3
I.2.1 Identification of GM-CSF……………………………………3
I.2.2 GM-CSF and T cells…………………………………….…...4
I.2.3 GM-CSF and tumor cell vaccine……………………….….....5
I.3 Regulatory T cell (Treg) …………………………………….…......6
I.3.1 Discovery and development…………………………….……6
I.3.2 Immunological features of Treg cells………………….…......7
I.3.3 Immunosuppressive function……………………………..….7
I.4 Specific Aims………………………………………………………8
CHAPTER II Materials and Methods………………………………...10
II.1 Cell lines…………………………………………………….10
II.2 Mice…………………………………………………………10
II.3 ELISA……………………………………………………….10
II.4 Vaccine immunization……………………………………….11
II.5 Isolation of lymphoid cells and splenocytes………………...12
II.6 Restimulation of lymphoid cells…………………………….12
II.7 Flow cytometry analysis…………………………………….12
CHAPTER III Results…………………………………………….......14
III.1 GM-CSF levels in TC-1, TC-1/wtGM, and TC-1/cGM cell
lines……………………………………………………….14
III.2 Schematic view of the experimental procedure of multiple
vaccinations……………………………………………….14
III.3 The increased splenic weight after high-dose GM-CSF
vaccine immunization………………………………….….15
III.4 The expression of regulatory T cells after multiple vaccinations with irradiated GM-CSF-secreting tumor cells……………………………………………………….16
III.5 The absolute numbers of regulatory T cells were consistent
with their expressions by flow cytometry…………………17
III.6 Multiple vaccinations with irradiated TC-1/cGM cells did not
augment the recall responses of E7-specific T cells………18
CHAPTER IV Discussion…………………………………………. .20
REFERENCE…………………………………………………………...25
Figures
Fig.1 GM-CSF levels in TC-1, TC-1/wtGM, and TC-1/cGM cell
lines………………………………………………………..35
Fig.2 Schematic view of the experimental procedure of multiple
vaccinations………………………………………………..36
Fig.3 The splenic weights of mice received multiple vaccinations……………………………………………..37
Fig.4 The expression of regulatory T cell in draining lymph nodes
after multiple vaccinations……………………………….. 38
Fig.5 The systemic immune responses of regulatory T cell after
multiple vaccinations…………………………………….. 40
Fig.6 The absolute number of Treg cell in lymphocytes and
splenocytes after multiple vaccinations…………………...42
Fig.7 Recall responses of HPV E7-specific T cells after multiple
vaccinations………………………………………………..43

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